Lysosomal Reacidification Ameliorates Vinyl Carbamate-Induced Toxicity and Disruption on Lysosomal pH

Article abstract of DOI:10.1021/acs.jafc.0c00534

Ethyl carbamate (EC) is a carcinogen toxicant, commonly found in fermented foods and beverages. The carcinogenic and toxic possibility of EC is thought to be related to its metabolite vinyl carbamate (VC). However, we found interesting mechanisms underlying VC-induced toxicity in this study, which were greatly different from EC. We first conducted a simple synthesis procedure for VC and found that VC possessed higher toxicity but failed to regulate levels of reactive oxygen species, glutathione, and autophagy. Notably, VC treatment resulted in upregulation of lysosomal pH, which was responsible for its cytotoxicity. Cyclic adenosine monophosphate (cAMP) pretreatment could enhance restoration of lysosomal acidity and ameliorate VC-induced damage. Inhibition of protein kinase A and cystic fibrosis transmembrane conductance regulator can block cAMP-induced cytoprotection. Together, our results provided the evidence for novel mechanisms of toxicity and possible protection method under VC exposure, which might give new perspectives on the study of EC-induced toxicity.

Full text of DOI:10.1021/acs.jafc.0c00534

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Article
Lysosomal Reacidification Ameliorates Vinyl Carbamate-Induced
Toxicity and Disruption on Lysosomal pH
Yuting Li, Dongwen Hu, Jifeng Qi, Sunliang Cui, and Wei Chen*
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ABSTRACT: Ethyl carbamate (EC) is a carcinogen toxicant, commonly found in fermented foods and beverages. The carcinogenic
and toxic possibility of EC is thought to be related to its metabolite vinyl carbamate (VC). However, we found interesting
mechanisms underlying VC-induced toxicity in this study, which were greatly different from EC. We first conducted a simple
synthesis procedure for VC and found that VC possessed higher toxicity but failed to regulate levels of reactive oxygen species,
glutathione, and autophagy. Notably, VC treatment resulted in upregulation of lysosomal pH, which was responsible for its
cytotoxicity. Cyclic adenosine monophosphate (cAMP) pretreatment could enhance restoration of lysosomal acidity and ameliorate
VC-induced damage. Inhibition of protein kinase A and cystic fibrosis transmembrane conductance regulator can block cAMP-
induced cytoprotection. Together, our results provided the evidence for novel mechanisms of toxicity and possible protection
method under VC exposure, which might give new perspectives on the study of EC-induced toxicity.
KEYWORDS: vinyl carbamate, lysosomal reacidification, cAMP, cytotoxicity
autophagy and other endocytic pathways, is involved in
recycling cellular waste by digesting macromolecules and
other materials.22,23 Hydrolytic enzymes within the lysosome
contribute to its multiple degradative functions and remain
active with highly acidic pH.24 Elevation in lysosomal pH,
induced by exogenous toxicants or endogenous malfunction,
can seriously damage activities of hydrolytic enzymes and
restrict the degradation in lysosomes, which ultimately leads to
cellular dysfunction and damage.25,26 The process of lysosomal
reacidification by treatment with exogenous reagents can
restore the acidity of lysosomes and ameliorate cellular
dysfunctions.25
Our study, therefore, is aimed at investigating the possible
mechanism of toxicity from VC exposure and potential
protective method for reducing VC-induced cellular damage.
We found that VC did not regulate the process of ROS
production and autophagy in L02 cells, which were greatly
different from its parent compound EC.17 Therefore, we tried
to explore other possible mechanisms and found that lysosome
alkalization might be the major reason for VC-induced
cytotoxicity. Lysosome reacidification by cyclic adenosine
monophosphate (cAMP) treatment could successfully rescue
the disruption of lysosomal pH and cytotoxicity under VC
exposure. We believe that our study may provide guidance for
further studies on hepatotoxicity induced by EC and VC
treatment.
1. INTRODUCTION
Ethyl carbamate (EC) is present naturally during processing of
fermented foods and beverages.1,2 The concentration of EC in
cigarette smoke is similar to that in foods.3 EC, which can lead
to development of lung, liver, pulmonary, gland, and skin
tumors,4−7 has been evaluated and classified as a probable
carcinogen to human (Group 2A) by International Agency for
Research on Cancer. Vinyl carbamate (VC) is a primary
metabolite of EC via catalysis by a cytochrome P450 enzyme
(CYP2E1) and found to possess higher carcinogenicity than
EC.8 VC exposure can cause higher levels of DNA adducts and
greater numbers of tumors in lungs compared with EC.7−9 EC
and VC are both used as the inducer of lung tumors in rodents
to investigate effective treatment on lung cancer and potential
mechanisms of lung carcinogenesis.10,11,12 In addition,
previous studies indicate the potential toxicity and carcinoge-
nicity caused by VC exposure in liver and small intestine.13,14
Therefore, the toxic and carcinogenic possibility of EC might
be related to its metabolite VC.8
Reactive oxygen species (ROS) have been shown to operate
as intracellular signaling molecules driving various essential
biochemical reactions.15 Excessive ROS generated by exoge-
nous toxicants, however, can damage normal cellular functions
via toxicity of oxygen.15,16 Our previous study found that redox
disturbance led to a significant loss of cell viability in human
normal hepatocyte L02 cells under EC exposure.17 High levels
of ROS can trigger the process of autophagy which serves as a
cellular defense pathway.18,19 Autophagy is an intracellular
waste disposal pathway maintaining normal functions of cells.20
The process of autophagy starts with the formation of
autophagosomes containing cytoplasmic compounds and
organelles, which are subsequently delivered to lysosome.21
Lysosome, as the terminal organelle in the process of
Received: January 22, 2020
Revised: July 25, 2020
Accepted: July 29, 2020
Published: July 29, 2020
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2. MATERIALS AND METHODS
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incubated with VX770, a CFTR potentiator, for 1 h and then treated
with 2.5 mM VC for 24 h. For the MTT assay, formazan precipitate
was dissolved in DMSO and detected at 490 nm after incubation with
MTT for 4 h. For the SRB assay, cells were subjected to fixation with
ice-cold trichloro acetic acid for 1 h and incubated with SRB solution
for 30 min. Then, cells were washed with 1% acetic acid for three
times. Remaining dye was dissolved in Tris solution and detected at
540 nm.
2.5. Determination of Intracellular ROS. Measurement of
intracellular ROS levels was conducted according to the previous
method with slight modifications.29,30 After treatment with different
concentrations (0, 0.625, 1.25, 2.5, 3.5, and 5 mM) of VC or (2.5, 5,
20, 40, 60, and 80 mM) of EC for 24 h, L02 cells were incubated with
10 μM DCFH-DA, a specific ROS fluorescence probe, for 30 min and
photographed under a fluorescence microscope. H2O2 (600 μM)
treatment were set as a positive control. The results were expressed as
mean DCF fluorescence intensity.
2.6. Cellular Glutathione Measurement. The glutathione
(GSH) concentration was detected using the GSH-specific probe
NDA, as described previously.31,32 After incubation with different
concentrations of VC or EC for 24 h, L02 cells were stained with 50
μM NDA for 30 min and imaged under a fluorescence microscope.
The results were expressed as mean NDA fluorescence intensity.
GSH/GSSG ratio was determined by via the GSH and GSSG assay kit
(Beyotime, China) based on the manufacturer’s instruction.
2.7. LysoTracker Red Staining. The effect of VC on lysosome
was determined using LysoTracker Red, as described previously with
some changes.33 L02 cells were treated with different concentrations
of VC for 24 h. Based on results of the MTT assay, time-dependent
changes of fluorescence intensity were tested at the concentration of
2.5 mM. To study the effect of CQ pretreatment on lysosome under
VC exposure, cells were treated with 2.5 mM VC for 24 h after
incubation with 20 μM CQ for 1 h. Then, L02 cells were incubated
with 100 nM LysoTracker Red for 30 min and subjected to
fluorescence microscope analysis. The results were expressed as mean
fluorescence intensity.
2.8. Estimation of Lysosomal pH Using LysoSensor Green.
The intralysosomal pH was detected using pH-sensitive probe
LysoSensor Green, according to the manufacturer’s instructions.
L02 cells were treated with VC (0, 0.625, 1.25, and 2.5 mM) for 24 h.
Effects of 2.5 mM VC treatment for different time (0, 3, 6, 9, 12, and
24 h) on LysoSensor Green fluorescence intensity were also tested.
To evaluate the effect of CQ pretreatment on lysosomal pH under VC
exposure, cells were treated with 20 μM CQ for 1 h and then with 2.5
mM VC for 24 h. To evaluate effect of cAMP-induced lysosomal
reacidification, cells were treated with 2.5 mM VC for 24 h, after
incubation with 4 mM cAMP with or without 3 μM H-89 or 3 μM
CFTRi for 1 h. To determine the role of CFTR in lysosomal
reacidification, L02 cells were first incubated with VX770 for 1 h,
followed by treatment with 2.5 mM VC for 24 h. Then, cells were
stained with 100 nM LysoSensor Green for 30 min and subjected to
fluorescence microscope analysis. The results were expressed as mean
fluorescence intensity.
2.1. Chemicals. Paraformaldehyde, dimethyl sulfoxide (DMSO),
chloroquine (CQ), 3-Methyladenine (3-MA), rapamycin (Rap),
trichloro acetic acid, Tris, sulforhodamine B (SRB), cAMP, H-89,
cystic fibrosis transmembrane conductance regulator (CFTR)
inhibitor 172 (CFTRi) and 3-(4,5-dimthyl-2-thiazolyl)-2,5-diphenyl-
2-H-tetrazolium bromide (MTT) were purchased from the Sigma-
Aldrich (St. Louis, MO, USA). LysoTracker Red and LysoSensor
Green were obtained from Thermo Fisher Scientific (Waltham, MA,
USA). Dichlorodihydrofluorescein diacetate (DCFH-DA) and
naphthalene-2,3-dicarboxal-dehyde (NDA) were obtained from
Molecular Probes, Inc. (Eugene, OR, USA). All other reagents used
were of analytical grade.
2.2. Synthesis of VC. Step 1. Iodobenzene (1 equiv weight, 1
equiv) and selenium powder (Se, 3 equiv) were added to a stirring
suspension of cuprous iodide (CuI, 0.1 equiv) in DMSO. The
reaction was stirred for 4 h. The reaction mixture was quenched with
distilled water and then filtrated. The filtrate was extracted with
EtOAc and brine in sequence. The combined organic layers were
dried over Na2SO4, filtered, evaporated, and purified by silica gel
column chromatography to give diphenyl diselenide (70% yield).
Step 2. Diphenyl diselenide (1 equiv) was dissolved in ethanol and
then NaBH (3 equiv) was added portion wise. The reaction was
stirred at room temperature for 15 min. After that, bromoethanol was
slowly added and stirred overnight. The reaction solution was
evaporated, re-dissolved in EtOAc, and then filtrated. The filtrate was
washed with brine for three times, and the combined organic layers
were dried over Na2SO4, filtered, evaporated, and purified by silica gel
column chromatography to give compound 1 (95% yield).
Step 3. Compound 1 (1 equiv) and potassium cyanate (2.5 equiv)
were dissolved in dichloromethane, and then trifluoroacetic acid (2
equiv) was slowly added to reaction solution and stirred overnight.
After that, the reaction mixture was quenched with distilled water and
then filtrated. The filtrate was extracted with dichloromethane and
brine in sequence. The combined organic layers were dried over
Na2SO4, filtered, evaporated, and purified by silica gel column
chromatography to give compound 2 (85% yield). Compound 2 (1
equiv) and m-CPBA (1 equiv) were dissolved in dichloromethane and
then stirred at room temperature overnight. After that, the reaction
solution was filtrated, and the filtrate was evaporated to give
compound 3 (80% yield). Compound 3 (1 equiv) and sodium
carbonate (5 equiv) were dissolved in THF and stirred for 3 h. The
reaction solution was filtrated. The filtrate was evaporated and
purified by silica gel column chromatography to give VC (78% yield).
The structure of synthesized VC was characterized by NMR. The
sample was dissolved in CDCl3. 1H and 13C NMR analyses were
performed on a Bruker AVANCE III spectrometer (14.1 T),
1
operating at Larmor frequencies of 600 MHz for H and 150 MHz
for 13C.
2.3. Cell Culture. Normal human hepatocyte L02 cell line was
purchased from the Type Culture Collection of Chinese Academy of
Sciences. Cells were cultured in RPMI 1640 medium with 10% fetal
bovine serum and incubated with an atmosphere of 5% CO2 at 37 °C.
The medium also contained 100 units/mL penicillin and 100 units/
mL streptomycin.
2.4. Cell Viability Detection. The cell viability under VC
exposure were determined using the MTT assay and SRB assay.27,28
L02 cells were incubated with different concentrations (0, 0.625, 1.25,
2.5, 3.5, 5, and 10 mM) of VC or (2.5, 5, 20, 40, 60, and 80 mM) of
EC for 24 h. Further study on cell viability under VC treatment was
tested at the concentration of 2.5 mM, which was close to the IC50
value. To analyze the role of autophagy in VC-treated L02 cells, cells
were incubated with Rap (1, 5, and 10 μM), 3-MA (0.5 mM) or CQ
(5, 10, and 20 μM) for 1 h, and then with 2.5 mM VC for 24 h. To
evaluate the effect of cAMP-induced lysosomal reacidification on VC-
induced cytotoxicity, cells were first treated with 4 mM cAMP with or
without 3 μM H-89 or 3 μM CFTRinh-172 (CFTRi) for 1 h and then
incubated with 2.5 mM VC for 24 h. To determine the effect of
CFTR on cell viability under VC exposure, L02 cells were first
2.9. GFP-RFP-LC3 Transfection Assay. L02 cells were trans-
fected with GFP-RFP-LC3 plasmid and X-tremeGENE HP DNA
transfection reagent (Roche, Switzerland) based on the manufac-
turer’s instruction. Then, cells were treated with different concen-
trations of VC for 24 h. For detection of effects of CQ and cAMP on
autophagy, cells were treated with 20 μM CQ or 4 mM cAMP for 1 h,
followed by incubation with 2.5 mM VC for 24 h. After that, cells
were observed under a fluorescence microscope.
2.10. Western Blot. Western bolt was conducted according to
previous studies with slight modifications.34,35 After treatment,
protein samples were extracted using RIPA lysis buffer. The protein
samples were subjected to gel electrophoresis. Then, the protein
samples were transferred to polyvinylidene fluoride membranes. Blots
were incubated in blocking buffer (10%, w/v, dried skimmed milk in
PBST) for 1 h and then with primary antibodies diluted in 5% bovine
serum albumin in PBST overnight at 4 °C. Anti-LC3 (Abcam,
ab48394, 1:2000), anti-LAMP-1 (Santa Cruz, sc-20011, 1:1000), anti-
B
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Figure 1. VC-induced cytotoxicity without affecting intracellular ROS. (A) Synthetic route of VC. (B,C) L02 cells were incubated with different
concentrations (0, 0.625, 1.25, 2.5, 5, and 10 mM) of VC or different concentrations (2.5, 5, 20, 40, 60, and 80 mM) of EC for 24 h. H2O2 (600
μM) was set as a negative control. The quantitative data of cell viability determined by MTT and SRB assays. (D) Effect of VC treatment on
intracellular ROS levels. (E) Quantitative data of panel (D) were calculated by ImageProPlus and expressed as mean DCF fluorescence intensity.
VC, vinyl carbamate; ROS, reactive oxygen species; and DCFH-DA, 2′,7′-dichlorofluorescin diacetate. *p < 0.05.
cathepsin B (Cell Signaling Technology, 31718, 1:1000), anti-
cathepsin D (Abcam, ab75852, 1:2000), and anti-GAPDH (Abcam,
ab181602, 1:10,000) antibodies were used as primary antibodies in
this study. After being washed with PBST, blots were further
incubated with secondary antibodies for 1 h. The secondary
antibodies were anti-rabbit and anti-mouse immunoglobulin B (Bio-
Rad, 170-6515 and 170-6516, 1:1000). Blots were imaged by
chemiluminescent HRP substrate (Millipore, USA).
2.11. Statistical Analysis. The value of IC50 was measured by
software SPSS 22.0. For detection of fluorescence intensity, five
randomized and independent microscopic fields were chosen for
calculation using Image-Pro Plus. Each selected field contained at
least 30 cells. Data were shown as means standard deviations (SD)
of at least three independent experiments. One-way analysis of
variance (Duncan test) conducted by software SPSS 22.0 was used for
statistical analyses. p < 0.05 was considered as statistical significance.
C
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Figure 2. CQ protected against VC-induced cytotoxicity. (A−C) After incubation with Rap, 3-MA, or CQ for 1 h, L02 cells were treated with 2.5
mM VC for 24 h. Effect of Rap, 3-MA, or CQ on cell viability under VC exposure by the MTT assay. (D) Effect of CQ on VC-induced cytotoxicity
tested by the SRB assay. (E) Immunoblot analysis of LC3 expression. (F) Morphology of L02 cells transfected with GFP-RFP-LC3. VC, vinyl
#
carbamate; CQ, chloroquine; Rap, rapamycin; and 3-MA, 3-methyladenine. p < 0.05 vs control group, *p < 0.05 vs VC group.
Figure 1. To confirm the structure of purified compound, LC−
MS/MS and NMR experiments were performed (Figure S1).
The results were listed as follows:
3. RESULTS
3.1. Synthesis of VC and Structure Elucidation. A
previous study has unveiled the possibility of hepatoxicity
caused by VC treatment.36 However, few studies have revealed
further mechanisms of hepatoxicity under VC exposure. We
first conducted a simple and effective synthesis of VC by
chemical oxidation of 2-(phenylseleno) ethanol using m-
CPBA. The detailed process of VC synthesis was shown in
1H NMR (600 M, CDCl3): δ 7.148 (dd, J1 = 13.8 Hz, J2 =
6.0 Hz, 1H), 4.769 (dd, J1 = 13.8 Hz, J2 = 1.8 Hz, 1H), 4.468
(dd, J1 = 6.6 Hz, J2 = 1.9 Hz, 1H).
13C NMR (150 M, CDCl3): δ 153.8, 142.0, 96.1.
3.2. VC-induced Cytotoxicity in L02 Cells. The effect of
VC treatment on hepatocyte L02 cells viability was
D
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Figure 3. Effect of CQ and VC treatment on lysosome. (A) L02 cells staining with LysoTracker Red after VC treatment for 24 h. (B) Quantitative
data of panel (A) were calculated by ImageProPlus and expressed as mean fluorescence intensity. (C) L02 cells staining with LysoTracker Red after
2.5 mM VC treatment for 3, 6, 9, 12, and 24 h. (D) Quantitative data of panel (C) were calculated by ImageProPlus and expressed as mean
fluorescence intensity. (E) Cells were pretreated with 20 μM CQ for 1 h and then incubated with 2.5 mM VC for 24 h. After treatment, L02 cells
were stained with LysoTracker Red. (F) Quantitative data of panel (E) were calculated by ImageProPlus and expressed as mean fluorescence
intensity. (G) Assessment of LAMP-1 expression by western blots. VC, vinyl carbamate. *p < 0.05.
subsequently evaluated by MTT and SRB assays. The MTT
assay showed that cell viability after VC exposure for 24 h
decreased dose-dependently (Figure 1B). A significant differ-
ence was not observed in 0.625 mM VC-treated group
compared with the control group (Figure 1B). The cell
viability decreased to 82, 50, 36, 30, and 19% under higher
concentrations (1.25, 2.5, 3.5, 5, and 10 mM) of VC treatment,
respectively. As a comparator, 40, 60, and 80 mM of EC
treatment also significantly reduced cell viability to 84, 78, and
48%, which was similar to the previous study.17 The IC50 value
for VC was 2.9 mM based on the MTT assay. Similar results of
the SRB assay were consistent with those of the MTT assay, as
shown in Figure 1C. Given these results, 2.5 mM VC was
chosen for following experiments. Hence, VC treatment could
decrease viability of L02 cells, and its toxicity was found to be
stronger than its parent compound EC.
3.3. VC Treatment did Not Regulate Levels of ROS
and GSH in L02 Cells. ROS overproduction, usually activated
under exposure of exogenous toxicants, can serve as an
important initiator for cytotoxicity.16 EC treatment was found
to trigger redox disturbance in different cell lines.17,37,38 As the
primary metabolite of EC, VC was also speculated to induce
intracellular ROS overproduction. Therefore, we measured
ROS levels in VC-treated L02 cells using DCFH-DA, a ROS-
specific fluorescence probe. Cells treated with 600 μM H2O2
were set as a positive control. H2O2 significantly increased
ROS levels by 49% compared with the control (100%). No
significant changes of fluorescence intensity were found after
VC exposure for 24 h, while 60 and 80 mM EC enhanced ROS
levels by 38 and 48%, respectively (Figure 1D,E). We also
examined the intracellular GSH concentration after VC
exposure. GSH serves as an essential antioxidant for
maintaining redox homeostasis. We observed that GSH levels
were markedly reduced to 77 and 58% when L02 cells were
exposed to 60 and 80 mM of EC. On the contrary, no
significant changes in GSH concentrations were found after
VC treatment (Figure S2A,B). Different concentrations of VC
did not affect the GSH/GSSG ratio, while 60 and 80 mM EC
treatment significantly decreased the GSH/GSSG ratio
compared with the control, which was consistent with results
of NDA staining (Figure S2C). Taken together, 2.5 mM VC
failed to affect intracellular ROS and GSH levels, which
suggested that VC might possess different toxic mechanisms
compared with EC.
3.4. VC Failed to Affect Autophagy in L02 Cells.
Autophagy, stimulated under endogenous and exogenous
cellular stress, serves as a cytoprotective system, which is an
important source of essential compounds for normal cellular
functions by lysosomal degradation of cytoplasmic materials
and damaged organelles.39,40 EC was found to promote the
process of autophagy previously.17 We, therefore, hypothesized
that the severe cytotoxicity caused by VC might also enhance
autophagy as a cell defense pathway. The MTT assay showed
that Rap (an inducer of autophagy) or 3-MA (an inhibitor of
autophagy) failed to affect cell viability compared with the VC-
treated group (Figure 2A,B). However, the pretreatment of 20
μM CQ, an autophagy inhibitor, contributed to an increase of
17% in cell viability compared with the VC group (Figure 2C).
E
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Figure 4. VC-induced lysosomal alkalization. (A) L02 cells were treated with different concentrations (0, 0.625, 1.25, and 2.5 mM) of VC for 24 h
and then stained with LysoSensor Green. (B) Quantitative data of panel (A) were calculated by ImageProPlus and expressed as mean fluorescence
intensity. (C) L02 cells were treated with 2.5 mM VC for different time (0, 3, 6, 9, 12, and 24 h) and then stained with LysoSensor Green. (D)
Quantitative data of panel (C) were calculated by ImageProPlus and expressed as mean fluorescence intensity. (E,F) Immunoblot analysis of CTSB
and CTSD expression. VC, vinyl carbamate; CQ, chloroquine; CTSB, cathepsin B; and CTSD, cathepsin D. *p < 0.05.
The results of SRB assays for CQ pretreatment were consistent
with those of MTT assays (Figure 2D). To further determine
whether VC treatment contributed to the regulation of
autophagy, we conducted western blot to examine levels of
LC3-II (a commonly used autophagosome marker). VC
treatment failed to induce any significant changes of LC3-II
levels (Figure 2E). We also conducted the GFP-RFP-LC3
transfection assay for further detection of autophagy flux. In
total, 0.625, 1.25, and 2.5 mM VC failed to affect number of
autolysosomes and autophagosomes (Figure 2F), which were
consistent with LC3-II expression. Together, VC exposure did
not affect the level of autophagy, which was also different from
the results of EC.
3.5. VC Treatment Caused Lysosomal Alkalization in
L02 Cells. The significant protection of CQ pretreatment
against VC-induced cytotoxicity could ascribe to the regulation
of lysosomal biogenesis and acidity.41−43 Hence, we speculated
that VC might affect lysosomes in L02 cells. The results of
LysoTracker Red staining assay showed that VC induced a
dose-dependent decrease in fluorescence intensity (Figure
3A,B). VC treatment with the concentration of 2.5 mM
significantly reduced fluorescence intensity by 15% (Figure
3A,B). Besides, a time-dependent decrease in fluorescence
intensity was also observed under the exposure of 2.5 mM VC
(Figure 3C,D). We also observed effect of CQ pretreatment on
fluorescence intensity of LysoTracker Red. As we expected,
CQ pretreatment significantly increased fluorescence intensity
compared with 2.5 mM VC-treated group, as shown in Figure
3E,F. Because CQ pretreatment could increase cell viability
compared with VC treatment, we speculated that VC-induced
toxicity might be related to the changes of lysosomes.
To further analyze the effect of VC exposure on the amount
of lysosome, we conducted western blot on the expression of
lysosomal-associated membrane protein 1 (LAMP-1). LAMP-
1, the most abundant lysosomal membrane protein accounting
for 50% of the total protein in the lysosomal membrane, is
used as the marker for detection of lysosome numbers.24 The
results of western blot showed that VC treatment with different
concentrations did not affect LAMP-1 expression (Figure 3G).
Then, we evaluated the changes of lysosomal pH under VC
treatment using a pH-sensitive fluorescence probe LysoSensor
Green. The results were similar to those of the LysoTracker
Red staining assay. Dose- and time-dependent decreases of
fluorescence intensity were detected under VC exposure
F
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Figure 5. cAMP promoted lysosomal re-acidification and rescued cytotoxicity caused by VC. After incubation with 4 mM cAMP or 20 μM CQ for
1 h, L02 cells were treated with 2.5 mM VC for 24 h. (A,C) L02 cells staining with LysoSensor Green. (B) Quantitative data of panel (A) were
calculated by ImageProPlus and expressed as mean fluorescence intensity. (D) Quantitative data of panel (C) were calculated by ImageProPlus and
expressed as mean fluorescence intensity. (E,F) Effect of cAMP on cell viability under VC exposure tested by MTT and SRB assays. VC, vinyl
carbamate; CQ, chloroquine; and cAMP, cyclic adenosine monophosphate. *p < 0.05.
(Figure 4A−D). Lysosomal acidity was crucial for lysosomal
function. Inactive precursors of cathepsins are synthesized in
cytoplasm and then cleaved to create the mature forms in
lysosomes, which were responsible for lysosomal degradation
function. 2.5 mM VC treatment also significantly inhibited the
expression of mature cathepsin B and D (CTSB and CTSD),
as shown in Figure 4E,F. Therefore, these results indicated that
VC treatment could disrupt lysosomal pH and cause lysosomal
dysfunction without affecting lysosome numbers in L02 cells.
3.6. CQ Activated Lysosomal Reacidification in VC-
Treated L02 Cells. CQ can induce a lasting lysosomal
reacidification in the previous study.41 Therefore, we
speculated that the protection of CQ pretreatment against
VC-induced cytotoxicity was ascribed to the restoration of
lysosomal acidity. CQ alone could cause higher fluorescence
intensity of LysoTracker Red than that of the control group,
which was consistent with the previous study.41 Similar results
are also shown in Figure 3E,F. Then, we confirmed our results
using pH-sensitive LysoSensor Green to evaluate changes of
lysosomal pH and observed similar results with LysoTracker
Red (Figure 5A,B). Taken together, CQ pretreatment could
activate lysosomal reacidification to protect against cytotoxicity
caused by VC.
3.7. cAMP Promoted Lysosomal Reacidification and
Reduced Cytotoxicity in L02 Cells. Despite the restoration
of lysosomal acidity under CQ treatment,41 CQ is also
reported to raise the pH of cellular compartments, especially
lysosomes.44 Given these discrepancies on alteration of
lysosomal pH by CQ treatment, we used cAMP, another
effective reagent for lysosomal reacidification,25,26 to restore
lysosomal pH in VC-treated cells. We found that cAMP
pretreatment could normalize lysosomal acidity of L02 cells
under VC exposure (Figure 5C,D). Treatment with cAMP
alone did not affect the basal acidity of lysosomes (Figure
5C,D), which was different from CQ exposure. Besides, we
found that mature CTSB and CTSD levels of cAMP + VC
groups were higher than the VC group, as shown in Figure
S3A,B, which might ascribe to the elevation of lysosomal
acidity. In addition, cAMP pretreatment significantly increased
cell viability by 16% compared with VC-treated group
according to the MTT assay (Figure 5E). SRB assays also
showed similar results in Figure 6F. Thus, cAMP pretreatment
not only promoted lysosomal reacidification but also sup-
pressed VC-induced cellular damage, which further confirmed
that lysosomal alkalization might be one of the main causes for
cytotoxicity in VC-treated L02 cells.
We also investigated CQ and cAMP pretreatment on
autophagy flux. Because CQ could inhibit autophagy flux and
cause autophagosomes accumulation, LC3-II level of the CQ
group was increased compared with the control (Figure S4A).
No significant changes of LC3-II expression were shown in the
CQ group and CQ + VC group, while cAMP pretreatment
increased LC3-II expression compared with the VC group
(Figure S4B). The GFP-RFP-LC3 transfection assay showed
similar results (Figure S4C). The red puncta and yellow puncta
represented autolysosomes and autophagosomes. The number
of yellow puncta in the CQ group was similar to that in the VC
+ CQ group, which indicated that VC failed to affect
autophagy flux. cAMP pretreatment significantly increased
the numbers of red puncta, which suggested that cAMP
pretreatment promoted autophagy flux. Based on these results,
we further detected cAMP-triggered lysosomal reacidification
in VC-treated cells.
3.8. PKA and CFTR Were Involved in Lysosomal
Reacidification Caused by cAMP. Protein kinase A (PKA),
a cAMP-dependent kinase, also has been found to promote
lysosomal reacidification.26 Therefore, we testified whether
PKA was involved in the process of lysosomal reacidification
caused by cAMP pretreatment in our study. The fluorescence
intensity of LysoSensor Green in L02 cells treated with the
PKA inhibitor H-89 alone was similar to that of the control
and cAMP group (Figure 6A,B). However, H-89 could weaken
the process of lysosomal reacidification caused by cAMP
(Figure 6A,B). We further tested the effect of H-89 on
increased cell viability under cAMP pretreatment. According to
MTT and SRB assays, we found that H-89 significantly
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Figure 6. PKA and CFTR were critical for cAMP-induced lysosomal reacidification. L02 cells were incubated with 4 mM cAMP with or without 3
μM H-89 or CFTRi for 1 h and then treated with 2.5 mM VC for 24 h. (A,E) L02 cells staining with LysoSensor Green. (B) Quantitative data of
panel (A) were calculated by ImageProPlus and expressed as mean fluorescence intensity. (F) Quantitative data of panel (E) were calculated by
ImageProPlus and expressed as mean fluorescence intensity. (C,D) Effect of H-89 and cAMP on VC-induced cytotoxicity tested by MTT and SRB
assays. (G,H) Effect of CFTRi and cAMP on VC-induced cytotoxicity tested by MTT and SRB assays. VC, vinyl carbamate; cAMP, cyclic
adenosine monophosphate; PKA, protein kinase A; and CFTRi, CFTR inhibitor 172. *p < 0.05.
inhibited the protection from cAMP against VC-induced
toxicity (Figure 6C,D). Therefore, PKA was involved in
lysosomal reacidification and cytoprotection caused by cAMP.
The restoration of lysosomal pH activated by cAMP/PKA is
usually related to CFTR, a PKA-mediated chloride channel
that enhances lysosomal reacidification by regulating the
exchanges of H+/Cl−.45 The effect of CFTR on cAMP/PKA-
induced pH restoration was further investigated in our study.
L02 cells treated with CFTRi, a CFTR inhibitor, alone showed
no significant changes in fluorescence intensity of LysoSensor
Green compared with the control and cAMP group (Figure
6E,F). As expected, CFTRi inhibited restoration of lysosomal
pH by cAMP pretreatment, which was similar to the results of
H-89 (Figure 6E,F). MTT and SRB assays also showed
significant reduction in cell viability caused by co-pretreatment
of CFTRi and cAMP, compared with pretreatment of cAMP
alone (Figure 6G,H). We further pretreated L02 cells with 5
μM VX770, a cAMP-independent CFTR potentiator,46 for 1 h
and followed by incubation with 2.5 mM VC for 24 h. VX770
pretreatment can significantly increase fluorescence intensity of
LysoSensor Green and cell viability, compared with cells
treated with VC alone (Figure S5A−D). These results,
therefore, indicated that CFTR also served as an essential
element for lysosomal reacidification.
4. DISCUSSION
In our study, we successfully synthesized VC, the primary
metabolite of EC, and provided novel findings related to VC-
induced cytotoxicity in human normal hepatocyte L02 cells.
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Despite its higher toxicity than EC, VC possesses greatly
different toxic mechanisms compared with its parent
compound. Interestingly, VC was found to induce lysosomal
alkalization, which further triggered severe cellular damage.
Lysosomal reacidification activated by cAMP pretreatment
could reduce VC-induced cytotoxicity. To the best of our
knowledge, this is the first study to elaborate the toxic
mechanisms and potential protection against toxicity under VC
exposure.
Our results first showed severe hepatoxicity of VC by
observing significantly decreased cell viability under VC
exposure in L02 cells. In our previous study, EC-induced
cytotoxicity was related to excessive ROS generation
accompanied with GSH depletion, and autophagy was
enhanced as a defense pathway.17 Conversely, VC treatment
failed to regulate the levels of ROS and GSH, as well as the
process of autophagy. The metabolism of EC is reported to
generate ethanol, ammonia, carbon dioxide, and VC.47 Ethanol
and ammonia have direct and indirect regulation on ROS
overproduction in the cytoplasm and mitochondria via the
disturbance of antioxidant pathways.48,49 Ethanol can also
trigger high levels of autophagy as a defense mechanism in
astroglia cells.50 Therefore, excess ROS generation and
enhancement of autophagy under EC exposure might ascribe
to other metabolites or EC itself.
with H-89 alone, a commonly used inhibitor of PKA, did not
alter basal pH in lysosomes, which is consistent with the
previous study.26 Co-pretreatment of H-89 with cAMP
blocked the process of cAMP-induced lysosomal reacidification
and protection against VC-induced cytotoxicity. The most
attractive explanation for improvement of lysosomal acidity by
the activation of cAMP/PKA would be through the enhance-
ment of CFTR. CFTR, a cAMP/PKA-mediated chloride
channel,45 appears to be crucial for the process of lysosomal
reacidification. The antagonist CFTRinh-172 (CFTRi) fail to
affect basic levels of lysosomal acidity but significantly reduced
the reacidification of compromised lysosomes under cAMP
pretreatment. In addition, the inhibition of CFTR also
suppressed cAMP-induced cytoprotection in VC-treated cells.
Thus, CFTR and PKA were critical regulators in the pathway
of cAMP-induced lysosomal reacidification and increased cell
viability in L02 cells.
These findings provided new perspectives on potential
mechanisms underlying hepatoxicity and possible protective
methods under treatment of EC, the parent compound of VC.
Besides VC, future studies should also focus on toxicity caused
by other metabolites for further explanation of EC-induced
damage.
ASSOCIATED CONTENT
■
Despite the absence of autophagy in VC-induced cytotox-
icity, we found CQ, a commonly used inhibitor for autophagy,
could prevent the decrease in cell viability under VC exposure.
CQ treatment has been found to induce a stable restoration of
the lysosomal acidity.41 Lysosome is the key degradative
compartment for metabolic processes including the recycling
of materials from organelles.51 The degradative functions of
lysosomes are potentiated by a profound luminal acidity.23
Disruption of lysosomal pH caused by stimuli is treated as the
main reason for cellular dysfunction and severe cytotoxicity.26
We, thus, speculated that VC could upregulate lysosomal pH,
and the protection from CQ against cytotoxicity might be
related to the restoration of lysosomal acidity. In this study, we
first found a significant decrease in fluorescence intensity of
LysoTracker Red under high levels of VC. CQ pretreatment
stimulated restoration of lysosomal pH by detecting increased
fluorescence intensity. This fluorescence dye will lose its
signals when the pH is over 6.5.50 LysoSensor, a more sensitive
fluorescence probe to pH, was used to further determine the
alteration of lysosomal pH. The results of LysoSensor Green
were in line with LysoTracker Red, which suggested that VC
treatment could lead to lysosomal alkalization, and lysosomal
reacidification induced by CQ could reduce its toxicity.
However, the effects of CQ treatment on lysosomal pH are
controversial in current literature, and the mechanism of CQ-
induced regulation on cellular metabolism and lysosomes was
still vague.41,42 Therefore, we used cAMP, another reagent
possessing the possibility of lysosomal reacidification, to
evaluate the role of lysosomal pH restoration in VC-induced
toxicity. Pretreatment with cAMP promoted the process of
lysosomal reacidification and remarkably rescued the cytotox-
icity induced by high levels of VC, which indicated that
lysosomal alkalization contributed to VC-induced cytotoxicity.
We therefore focused on the role of lysosomal reacidification
as a possible therapeutic method for cellular damage caused by
VC.
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c00534.
1H NMR and 13C NMR for VC; L02 cells treated with
different concentrations of VC or EC; and L02 cells
incubated with 4 mM cAMP, 20 μM CQ, and 5 μM
VX770 and then treated with 2.5 mM VC (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Wei Chen − Department of Food Science and Nutrition,
Zhejiang Key Laboratory for Agro-Food Processing and Ningbo
Research Institute, Zhejiang University, Hangzhou 310058,
China; orcid.org/0000-0002-2373-2437; Phone: +86 571
88982861; Email: zjuchenwei@zju.edu.cn; Fax: +86 571
88982191
Authors
Yuting Li − Department of Food Science and Nutrition, Zhejiang
Key Laboratory for Agro-Food Processing, Zhejiang University,
Hangzhou 310058, China
Dongwen Hu − Department of Food Science and Nutrition,
Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang
University, Hangzhou 310058, China
Jifeng Qi − Institute of Drug Discovery and Design, College of
Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, China
Sunliang Cui − Institute of Drug Discovery and Design, College
of Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c00534
Notes
The mechanism of lysosomal reacidification of cAMP might
be related to PKA, a kinase activated by cAMP.52 Treatment
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by grants from the Zhejiang
Provincial Natural Science Foundation of China
(LR18C200002) and the National Natural Science Foundation
of China (21876152).
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